CD74 induces TAp63 expression leading to B-cell survival by Frida Lantner, Diana Starlets, Yael Gore, Liat Flaishon, Ayala Yamit-Hezi, Rivka Dikstein, Lin Leng, Richard Bucala, Yossy Machluf, Moshe Oren, and Idit Shachar Blood Volume 110(13):4303-4311 December 15, 2007 ©2007 by American Society of Hematology

Diagram of tagged CD74 constructs used in this study. Frida Lantner et al. Blood 2007;110:4303-4311 ©2007 by American Society of Hematology

CD74 up-regulates TAp63 α and γ expression. CD74 up-regulates TAp63 α and γ expression. (A) HEK-293 cells were transfected with GFP or CD74-GFP constructs for 20 hours. Total RNA was isolated and reverse transcription was carried out using Superscript II RT. RT-PCR was performed to determine p63, TAp63, ΔNP63, TAp63α, and TAp63γ mRNA levels. The results presented are representative of 3 to 10 experiments. (B) HEK-293 cells were transfected with GFP or CD74-GFP constructs for 20 hours. Cells were collected and lysed as described in “Cell lysis by hot SDS,” and lysates were separated on 5% to 15% (wt/vol) gradient SDS-PAGE and blotted with anti-p63 antibody followed by HRP-conjugated antirabbit antibodies. The arrows indicate bands of 80 and 56 kDa, representing p63TAα and p63TAγ. The results presented are representative of at least 4 separate experiments. The intensity of the p63 band was divided by the intensity of the actin or tubulin band. The activation fold ratio in the GFP-transfected cells was normalized to 1, and the ratio for each transcription was calculated as the intensity of the sample, relative to 1. Frida Lantner et al. Blood 2007;110:4303-4311 ©2007 by American Society of Hematology

Translocation of CD74-ICD to the nucleus is required for TAp63 transcription. Translocation of CD74-ICD to the nucleus is required for TAp63 transcription. (A) HEK-293 cells were transfected for 20 hours with various constructs of CD74. Transfection efficiency was determined by analyzing the mRNA levels of a sequence that appears in all CD74 constructs. RNA was isolated and TAp63 transcription was determined by RT-PCR. The results presented are representative of 3 different experiments. (B) HEK-293 cells were transfected with empty vector or a construct containing CD74-ICD conjugated to a nuclear localization signal (1–42 nuc). Total RNA was extracted and real-time PCR was performed as described in “Real-time reverse-transciption–PCR analysis.” mRNA levels are shown after normalization for the HPRT control. Error bars represent SD. (C,D) HEK-293 cells were transfected with empty vector or a construct containing CD74-ICD conjugated to a nuclear localization signal (1-42 nuc). TAp63α and γ transcription was followed by RT-PCR. The results presented are representative of 8 independent experiments (C). Western blot showing protein expression of p63 isotypes. Cells were collected and lysed as described in “Cell lysis by hot SDS,” and lysates were separated on a 5% to 15% (wt/vol) gradient SDS-PAGE and blotted with anti-p63 antibody, followed by HRP-conjugated antirabbit antibodies. Arrows indicate bands of 80 and 56 kDa, representing p63TAα and p63TAγ. The results presented are representative of 5 different experiments (D). The intensity of the p63 band was calculated as described in Figure 2. Frida Lantner et al. Blood 2007;110:4303-4311 ©2007 by American Society of Hematology

Activation of CD74 expressed on primary B cells controls TAp63 transcription. Activation of CD74 expressed on primary B cells controls TAp63 transcription. (A) Primary B cells were transfected for 8 hours with various constructs of CD74 using AMAXA reagents, as described in “Cell transfection.” Transfection efficiency was determined by analyzing the mRNA levels of a sequence that is shared in all CD74 constructs. RNA was isolated and TAp63 transcription was determined by RT-PCR. (B) Control IgD+ or CD74−/− B cells were incubated in the presence or absence of anti-CD74 antibody or control anti-CD8 antibody for 24 hours. Total RNA was isolated and RT-PCR was performed, as described in “RNA isolation and reverse transciption.” The results presented are representative of 3 different experiments. (C) Control IgD+ cells were incubated in the presence or absence of anti-CD74 antibody, for various lengths of time. Total RNA was isolated and RT-PCR was performed, as described in “RNA isolation and reverse transciption.” The results presented are representative of 3 different experiments. (D) Primary B cells were incubated with MIF 100 μg/mL for various lengths of time. Total RNA was isolated and RT-PCR was performed, as described in “RNA isolation and reverse transcription.” The results presented are representative of 3 different experiments. The intensity of the p63 band was calculated as described in Figure 2. Frida Lantner et al. Blood 2007;110:4303-4311 ©2007 by American Society of Hematology

CD74 induces activation of the p65 member of the NF-κB family, which in turn activates TAp63 transcription. CD74 induces activation of the p65 member of the NF-κB family, which in turn activates TAp63 transcription. HEK-293 cells were transfected with empty, p65, or p65 + p50 constructs for 20 hours. (A) Analysis of HEK-293 cells transfected with NF-κB proteins by electrophoresis mobility shift assay using the NF-κB–binding site as DNA probe. (B) Nuclear extracts from p65-, p65 + p50–, and p50-transfected cells were used for DNA-binding reactions as indicated at the top the lanes. Total RNA was isolated, and RT-PCR was performed as described in “RNA isolation and reverse transciption.” (C) Cells were then collected and lysed, as described in “Cell lysis by hot SDS.” Lysates were separated on 5% to 15% (wt/vol) gradient SDS-PAGE, and blotted with anti-p63 antibody, followed by HRP-conjugated antirabbit antibodies. Arrows indicate bands of 80 and 56 kDa, representing p63TAα and p63TAγ. (D) Schematic representation of the p63 promoters. The sequences indicated are the PCR primers used for generating the luciferase constructs. (E,F) TAp63 activation was analyzed by luciferase assay, as described in “Luciferase assay for monitoring p63 activation.” (E) The HEK-293 cells were transfected with a luciferase construct containing the TAp63 promoter, together with a p65 construct or CD74-ICD (aa's 1-42), in the absence or presence of IκB. Cells were lysed, and luciferase activity was measured. Luciferase activities were normalized to the activity of the cotransfected RSV promoter–driven Renilla reporter luciferase, which was used to correct for differences in transfection efficiencies. Fold activation was calculated as the activity of p65 or CD74 constructs relative to the activity of the empty plasmid. The results presented are representative of 3 different experiments. (F) HEK-293 cells were transfected for 20 hours with luciferase constructs containing the TAp63 or ΔNp63 promoters, together with CD74-ICD (aa 1-42's). Cells were then lysed and luciferase activities were normalized to the activity of cotransfected RSV promoter–driven Renilla reporter luciferase, which was used to correct for differences in transfection efficiencies. Fold activation was calculated as the activity of the CD74-ICD construct, relative to the activity of cells transfected with an empty plasmid. The graph represents the average of 5 independent experiments. The results presented are representative of 3 different experiments. The intensity of the p63 band was calculated as described in Figure 2. Error bars represent SD. Frida Lantner et al. Blood 2007;110:4303-4311 ©2007 by American Society of Hematology

TAp63 regulates Bcl-2 expression. TAp63 regulates Bcl-2 expression. (A-B) HEK-293 cells were transfected with empty, p63TAα, and p63TAγ constructs for 24 hours. (A) Total RNA was isolated, and RT-PCR for Bcl-2 was performed as described in “RNA isolation and reverse transciption.” The results presented are representative of 4 different experiments. (B) Cells were collected and lysed as described in “Cell lysis by hot SDS,” and lysates were separated on 10% (wt/vol) SDS-PAGE and blotted with anti–Bcl-2 antibody, followed by HRP-conjugated antimouse antibodies. The membrane was stripped and reblotted with antitubulin. The arrow indicates the Bcl-2 band. The results presented are representative of 3 separate experiments. (C) ChIP analysis of p63 binding to the Bcl-2 promoter. HEK-293 cells were transfected with empty, p63TAα, and p63TAγ constructs for 24 hours. Chromatin prepared from these cells was immunoprecipitated with control and anti-p63 antibodies. Presence of the promoter sequence was then quantified by RT-PCR. (D) Primary B cells were transfected for 8 hours with various constructs of p63 using AMAXA reagents, as described in “Cell transfection.” RNA was isolated and Bcl-2 transcription was determined by RT-PCR. The results presented are representative of 3 different experiments. (E-H) siRNA for p63 or lacZ (control) was transfected into primary B cells in the presence or absence of anti-CD74 (E) or MIF (G,H) stimulation. Total RNA was isolated, and RT-PCR was performed as described in “RNA isolation and reverse transciption.” The results presented are representative of 4 different experiments (E,G). Annexin staining of B220+ cells was performed (F). Annexin and PI staining of B220+ cells was performed (H). The intensity of the p63 band was calculated as described in Figure 2. Numbers on the plots in panel H are percentages of total cells. Frida Lantner et al. Blood 2007;110:4303-4311 ©2007 by American Society of Hematology

p63 regulates B-cell survival. p63 regulates B-cell survival. (A-C) DNA screening of the wild-type (+/+), heterozygotic (+/−), and p63−/− (−/−) fetal liver cells (as previously described12) that were injected into irradiated mice (A). Splenocytes derived from the chimeric mice were stimulated with or without anti-CD74, and TAp63 or Bcl-2 mRNA levels were determined by RT-PCR (B). Annexin staining was performed on freshly isolated splenocytes (time 0) and on cells cultured for 12 or 24 hours. Histograms show the annexin-positive population of B220+ cells. The results presented are representative of 4 different experiments (C). (D) Primary CD74−/− B cells were transfected with empty plasmid, CD74-ICD, TAp63α, TAp63γ, or ΔNp63 constructs as described in “Cell transfection.” After 48 hours, the IgD+ population was analyzed by FACS. The results presented are the mean of 3 independent experiments. Error bars represent SD. The percentage increase of IgD+ cells was calculated by subtracting staining of empty expression plasmid from the percentage of IgD+ cells in each treatment, and dividing it into the same increased value, multiplied by 100%. The results presented are representative of 5 different experiments. The intensity of the p63 band was calculated as described in Figure 2. Frida Lantner et al. Blood 2007;110:4303-4311 ©2007 by American Society of Hematology